Process for the preparation of alpha-lglutamyl-choline and its salts



United States Patent 3,009,923 PROCESS FOR THE PREPARATION OF oz-L-GLUTAMYL-CHOLINE AND ITS SALTS Gaston Arniard, Noisy-le-Sec, and ReneHeymes, Romainville, France, assignors to UCLAF, Paris, France, acorporation of France No Drawing. Filed Nov. 20, 1959, Ser. No. 854,245Claims priority, application France Dec. 15, 1958. 12 Claims. (Cl.260-389) The present invention is directed to a process for thepreparation of a-L-glutamyl-choline and its salts substantially free ofits 'y-isomer. US. Patent No. 2,776,923 describes a process for thepreparation of L-glutamyl choline and its hydrochloride by the reactionof N-carbobenzoxy-L-glutamic acid anhydride with choline or thehydrochloride of choline. A mixture of the uand 'y-iso-. mers ofN-carbobenzoxylated L-glutamyl-choline or its hydrochloride is obtainedthereby. Hydrogenation in the presence of platinum orpalladium yields amixture of the 12- and. 'y-isorners of L-glutamyl-choline or itshydrochloride. v

A process which makes it possible to obtain prefer'enti-I ally theu-isomer of L-glutamyl-choline and its salts substantially free of its'y-is omer has heretofore not been known, and it is an object of thepresent invention to remedy this deficiency. a

Another object of this invention is the obtention of the non-toxicnitrate and iodide of a-L-glutamyl-choline substantially free of their'y-isomers.

A further object of this invention is the isolation of variousintermediate compounds useful in preparing a-L- glutamyl-choline salts.

These and further objects of the invention will become more apparent asthe description of the invention proceeds.

Q ,We have discovered that a-L-glutamyl-choline salts substantially freeof their 'y-isomers can be prepared by the following process.Glycol-monochlorohydrin is reacted at elevated temperatures withL-glutamic acid in the presence ofv gaseous hydrochloric acid, thehydrochloride ,of di-(BGchloroethyl) L-glutamate is isolated. Thishydrochloride is treated with an appropriate strong organic or inorganicbase, such as triethylamine or an alkali metal hydroxide, preferablysodium hydroxide, to obtain the free di-(,6-chloroethyl) L-glutamate.The free amine is 'tritylated, preferably by reacting with tritylchloride in the presence of a tertiary amine, such as triethyla rnineand the di- (,8'-chloroethyl) N-trityl-L-glutamate is isolated. Thiscompound is dissolved in methanol and reacted with sodium methylate togive a methanolysis of the-fy-carboxyl group. The a-(fi-chloroethyl)-'-methy1 ester of N-trityl- L-gl utamic acid is isolated. This mixedester is dissolved in an inert organic solvent and partially saponifiedwith an alkali metal hydroxide such as sodium hydroxide in dioxansolution. The (Z-(fi'-ChlOlOthy1) N-trityl-L-glutamate ester is isolatedeither in the form of an amine salt of its acid, such as thediethylamine salt, or in the form of the free acid. The free acid istreated with an alkali metal iodide such. assodium iodide andtrimethylamine in an anhydrous organic solvent such as acetone in. aclosed .vessel'at elevated temperatures The choline derivative is thendetritylated preferably by treating with an aqueous lower alka'noic acidsuch .as' 50% aqueous acetic acid and the iodide of aL-glutarnyl-choline is isolated. The iodide is transformed into thenitrate by the action of silvernitrate inaqueous solution on the iodideof ix-L-glutamylcholine. Other salts can be produced'by reaction with anappropriate reactant.

Patented Nov. 21, 1961 The series of reactions described above are shownin the following table:

N 0 (com).

X=a non-toxic anion As indicated in US. Patent 2,776,923,L-glutarnyl-choline and its salts are products exhibiting an interestingpharmacodyna-mic activity; they have been recommended for use in thetreatment of poliomyelitis and hypertension and as growth promotingagents.

According to Yoshito Nishizawa, Archives Francaises de Pediatric, 15,195-216 (1958), a-L-glutamyl-choline has twice the therapeutic activityof 'y-L-glutamyl-choline.

The following examples illustrate the invention. It is to be understood,however, that the invention is not limited'to the examples and suchchanges and modifications as occur to one skilled in the art arecontemplated in the practice of the invention. J

Example I.Prp.arati0n of the hydrochloride of di-(fl chloroethyl)L-glutamate (II) 20 gm. of L-glutamic acid (I) are introduced into'ZOOcc. of glycol-monochlorohydrin, and the mixture is heated on an oil bathat 15 C., while agitating and passing gaseous hydrochloric acidtherethrough until the mixture is saturated and completely dissolved.This requires approximately 30 minutes.

The solution is allowed to stand at the same temperature for 3 hoursmore and then distilled on a water bath under'reduced pressure untilabout 150 cc. of glycol-monochlorohydrin pass over, 250 cc. of ether areadded to the residue, the mixture is iced and filtered on a vacuumfilter. The filter cake is washed with ether by trituration and dried at50 (3., yielding 39 gm. (93%) of the hydrochloride ofdi-(/3-chloroethyl) L-glutamate (11) having a melting point of to 117C,and an optical rotation [a] =;l-16:0.5 (c.=2% in water). This product,which has not previously been described, is obtained in the form ofcolorless leafiets'which are soluble in water and alcohol, slightlysoluble in acetone and chloroform, and insoluble in ether. 1 1

, Analysis.C H O NCl mol. wt. 308.6. Calculated: C, 35.02%; H, 5.22%; O,20.73%; N,'4.54%; Cl, 34.47%. Found: C, 35.3%; H, 5.3%; O, 20.6%; N,4.5%; CI, 34.2%. 1

3 Example II.-'Preparati0n of a-(fl'-chloroethyl)-'y-methylN-trityl-L-glutamme (IV in which R=CH (a) DI-(B'-CHLOROETHYL L-GLUTAMATE31 gm. of di-(B-chloroethyl) L-glutamate hydrochloride, obtained inaccordance with the preceding example, are introduced into 50 cc. of icewater; thereafter, 250 cc. of chloroform, 2 drops of phenolphthalein andthen, in small portions, 12.5 cc. of concentrated sodium hydroxidesolution are added. The chloroform phase is decanted, the aqueoussolution is extracted with 25 cc. of chloroform, the chloroformsolutions are combined, washed with 15 cc. of water and dried overmagnesium sulfate.

(1)) DI-(B'-CHLOROETHYL) N-TRITYL-L-GLUTAMATE (III) i 30 gm. of tritylchloride are added to the chloroform solution obtained in (a) above, andthereafter 15 cc. of triethylamine are added in small portions. Themixture is allowed to stand overnight at 30 C. It is then washed with 50cc. of water, with a mixture consisting of 50 cc.

of water and 45 cc. of 1 Nhydrochloric acid and, finally, with 40 cc. ofwater, dried over magnesium sulfate and evaporated in vacuo. The residueobtained is primarily di-(pY-chloroethyl) N-trityl-L-glutamate.

(c) a-(B-CHLOROETHYL)-'y-METHYL N-TRITYL-L GLUTAMATE (IV IN -WHICHR=CHs) The residue obtained in ('b) above is dissolved in 50 cc. ofmethanol, evaporated to dryness in vacuo and taken up in 200 cc. ofmethanol at 35 C. 2.5 cc. of a 1 N solution I of sodium methylate inmethanol are added and the mixture is allowed to stand for 20 minutes at35 C. 1 cc. more of the sodium methylate solution is added and after 40minutes of standing, another 1 cc. is added. The mixture is allowed tostand at 35 C. forone hour and at room temperature for one hour.Thereafter, it is iced, filtered on a vacuum filter, the filter cakerinsed with iced methanol and dried at 40 C. 30 gm. of the raw productare obtained, this product is taken up in 50 cc. of carbontetrachloride. The solution is iced and the insoluble matter is filteredoff. The'filtrate is concentrated to onehalf its volume, 50 .cc. ofmethanol are added and the remaining carbon tetrachlorideis distilledoff. The solution is iced, filtered on ;a vacuum ,filter, thefilter cakeis rinsed with iced methanol and dried at 40 C. 29.2 gm. 63%) of a-(fichloroethyl) emethyl Ntrityl-L-glutamate (IV in which R=CH having amelting point of 83-86" C. and an optical rotation [al =+35.5i0.5'(c.=2% in chloroform) are obtained.

Analysis.C I-I O NCl; mohwt. 465 .95. Calculated: .C, 69.59%; 6.05%; N,3.0%; Cl, 7.61%. Found: C, 69.8%; H, 6.0%; N, 3.1%; Cl, 7.8%.

Example lIL-Preparation of a-(p chloroethyl) N-trityl-Leglu'tamate (IVinwhich R=--'-H) 4.7 gm. of a-(,8'-chloroethyl)-'y-methyl N-trityl-L-glutamate, obtained according to the preceding example :are dissolved in40 cc. of dioxan, and to this solution 10 cc. of methanol and 10.5 cc.of 1 N sodiumhydroxide are added. The mixture is placed on an ice waterbath and allowed to stand overnight while warming to room temperature.

The major portion of the solvents are evaporated in vacuo. 50 cc. ofwater are added and-the distillation of the dioxan is completed. 40 cc.of chloroform is added to the aqueous solution and thereafter 1 1 cc. of1 'N'hydrochloric acid are added. The mixture is agitated, thendecanted, and extracted twice with 5 cc. portions of chloroform. Thechloroform solutions are combined, washed with cc. of water, dried overmagnesium sulfate and evaporated to dryness in vacuo. a-(E'chloroethyl)N-trityl-L- glutamate IV'in which R= H) is obtained in a quantitativeyield.

This substance, which is obtained in the form of an oil,

has not previously been described; it may be used as such in Example V.

Example IV.-Characterizati0n of a-(p chloroethyl) N-zrityl-L-glutamate(IV in which R=-H) in the form of its '7 diethylamine salt The raw oilobtained in the preceding example is taken up in 10 cc. of ether. 1.1cc. of diethylamine are added and then, in two portions, 20 cc.cyclohexane are added. The mixture is iced for'2 hours and filtered on avacuum filter. The filter cake is washed with cyclohexane and dried. 4.7gm. of the 'y-diethylaminesalt of .oc(]3'-Chl010- ethyl) N-tritylbglutamate, having a melting point around C. and an optical rotation [u]=+46i (c.=*2% in chloroform), are obtained. This product, which isnovel, is obtained in the form of colorless small needles, which aresoluble in water, alcohol, acetone, benzene and chloroform, and slightlysoluble in ether and cyclohexane.

Analysis.-C H O N Cl; mol. Wt. 525.077. Calculated: C, 68.62%; H, 7.1%;N, 5.33%; Cl, 6.75%. Found: C, 68.8%; H, 7.1%; N, 5.1%; CI, 6.2%.

Example V.--Preparati0n of the iodide of a-L-glummylcholine (V) in whichX =1) (a) a-(B'-CHLOROETHYL) N-TRITYL-L-GLUTAMATE (IV IN WHICH R=-H) gm.of the -y-diethylamine salt of a-(fi-chloroethyl) N-trityl-L-glutamateareintroduced into 100 cc. of chloroform, 20 cc. of water are added andthen in small portions and while agitating, 30 cc. of 1 N hydrochloricacid are added. The aqueous phase is decanted and extracted with 20 cc.of chloroform. The chloroform extracts are combined, washed with 30 cc.of water,'dried over magnesiumsulfate and evaporated to dryness invacuo.

(b) IODIDE OF THE .a-CHOLINE-N-TRITYL- L-GLUTAMATE 6 gm. of'moltensodium iodide and then a solution of the u-(B'-chloroethyl)N-trityl-L-glutamate obtained under (a) above in 20 cc. of dry acetoneare introduced into a scalable tube. 12 cc. of anhydrous tr'imethylamineare added, the tube issealed and the contents are heated overnight at 75C. The tube is iced and opened, the contents are removed and evaporatedto dryness in vacuo. The residue is taken up in 60 cc. of chloroform andthe insoluble matter is filtered off. '20 cc. of water containing 1 cc..ofacetic acid are added to the filtered solution. The solution isacidified to a pl-I of '2 with a dilute aqueous solution of hydroiodicacid. The aqueous phase is decanted and extracted with chloroform. Thechloroform extracts are washed with 10 cc. of water and dried overmagnesium sulfate. The extract is evaporated to dryness in vacuo. 120cc. of anhydrous ether are added, the mixture is stirred, filtered andthe filter cake is dried in vacuo. 10.7 gm. of a raw product are thusobtained. This product is pulverized and .again treated with ether aspreviously described in order to obtain 9.8 gm. (54%) of the iodide ofa-choline ester of N-trityl-L-glutamic acid.

.(c) PREPARATION OF THE IODIDE OF a-L-GLUTAMYL- CHOLINE (V- IN WHICHX=I) 7.2 cc. of a 50% aqueous solution of acetic acid are added to 3 gm.of the raw iodide of the wcholine ester of N-trityl-L-glutamic acidobtained in (b) above, the mixture is stirred and heated at 35 ,C. for1,5 minutes. 118 cc. of acetone and then, in portions, 25 cc. of acetoneare added. The mixture is filtered on a vacuum filter, the filter cakeis Washed with acetone and dried in vacuo to obtain 1.35 gm. (77%) ofthe iodide 0f .wLlglutamylcholine, having a melting point of to C. andan optical rotation [a] =+13.5- *-1 .(c.=2% in water). This product,which has not previously been described, isobtained in the form of smallcolorless needles which are soluble in water and insoluble in alcohol,ether, acetone and benzene.

Analysis.-C H O N l; mol. Wt. 360.21. Calculated: C, 33.34%; H, 5.88%;N, 7.77%; I, 35.24%. Found: C, 33.3%, H, 5.8%; N, 7.7%; I, 36.0%.

Example VI.Preparatin 0f the nitrate 0f a-L-glutamyl choline (V in whichX NO 1.08 gm. of the iodide of u-L-glutamyl choline obtained inaccordance with the preceding example are introduced into a solution of0.510 gm. of silver nitrate in 4 cc. of water. The mixture is filtered,concentrated to /2 its volume, 4 cc. of alcohol and 40 cc. of acetoneare added, and the mixture is iced and filtered on a vacuum filter. Thefilter cake is recrystallized from 2 cc. of water containing a smallamount of alcohol by addition of acetone in order to obtain 650 mgm.(75%) of the nitrate of a-L-glutamyl choline having a melting point ofabout 90 C. (in a capillary tube) and an optical rotation [a] =16.5i1(c.=2% in Water).

This product, which is new, is obtained in the form of small colorlessleaflets which are soluble in water, slightly soluble in alcohol andinsoluble in ether, acetone and benzene.

Analysis.--C H O N mol. wt. 259.29. Calculated: C, 40.67%; H, 7.17%; N,14.23%. Found: C, 40.6%, H, 7.2%; N, 13.9%.

It will be understood that the invention is not limited to the processdescribed in the preceding examples nor to the products thus obtained.More particularly, the

nature of the solvents and reactants, as well as the reactiontemperatures may be varied without departing from the scope of theinvention. It is readily apparent to those skilled in the art thatvarious changes and modifications may be made without departing from thespirit of the invention or the scope of the appended claims.

We claim:

1. Non-toxic salts of a-L-glutamyLcholine substantially free of their'y-isomers selected from the group consisting of the iodide ofa-L-glutamyl-choline and the nitrate of a-L-glutamyl-choline.

2. The iodide of a-L-glutamyl-choline substantially free of its'y-isomer.

3. The nitrate of a-L-glutamyl-choline substantially free of its'y-isomer.

4. a-(B'-Chloroetl1yl) -'y-methy1 N-trityl-L-glutamate.

5. a-(e'-hloroethyl) ester of N-trityl-L-glutamate.

6. The iodide of the a-cho'line ester of N-trityl-L-glutamic acid.

7. A process for the production of a non-toxic Salt ofa-L-glutamyl-choline substantially free of its q-isomer comprising thesteps of (a) esterifying L-glutamic acid with glycol-monochlorohydrin inthe presence of gaseous hydrochloric acid at elevated temperatures, (b)neutralizing the hydrochloride of di-(fi'chloroethyl) L-glutamate bytreatment with a strong base selected from the group consisting oftriethylamine and alkali metal hydroxides, (c) reacting thedi-(}9'-chloroethyl) L-glutamate with trityl chloride in the presence ofa tertiary amine, (d) dissolving the di-(H-chloroethyl) N-trityl-L-glutamate in methanol and reacting with sodium methylate, (e) dissolvingthe a-(echlomethyl) y-methyl N-hityl-L-glutamate in an inert organicsolvent and saponifying with an alkali metal hydroxide, (f) quaternizingthe cc-(B'-Chl0l'0- ethyl) ester of N-trityl-L-glutamic acid by reactingwith trimethylamine and an alkali metal iodide in an anhydrous organicsolvent under pressure at elevated temperatures, (g) detritylating theiodide of the DL-ChOlll'lfi ester of N-trityl-L-glutamic acid bycontacting with an aqueous lower alkanoic acid, (h) and isolating anon-toxic salt of a-L-glut-amyl choline substantially free of its-isomer.

8. The process of claim 7 wherein the strong base used in theneutralizing step b is sodium hydroxide.

9. The process of claim 7 wherein the tertiary amine used in thereacting step c is triethy-lamine.

10. The process of claim 7 wherein the inert organic solvent and thealkali metal hydroxide used in the dissolving and saponifying step e arerespectively diox-an and sodium hydroxide.

11. The process of claim 7 wherein the aqueous lower alkanoic acid usedin the detritylating step g is a 50% aqueous solution of acetic acid.

12. A process for the production of a non-toxic salt ofa-L-glutamyl-choline substantially free of its 'y-isomer comprising thesteps of (a) esterifying L-glutamic acid with glycol-monochlorohydrin inthe presence of gaseous hydrochloric acid at a temperature between aboutC. and C., (b) neutralizing the hydrochloride of di- (fi-chloroethyl)L-glutamate by treatment with a solution of an alkali metal hydroxide,(c) reacting the di- (K-chloroethyl) L-glutamate with trityl chloride inthe presence of triethylamine, (d) dissolving the di-(fl'-chloroethyi)N-trityl-L-glutamate in methanol and reacting withsodium methylate, (e)dissolving the cc-(fi'-Chl0i0- ethyl)-' -methyl N-trityl-L-glutamate indioxan and saponifying with an alkali metal hydroxide, (f) quaternizingthe a-(fi-chlor'oethyl) ester of N-trityl-L-glutarnic acid by reactingwith trimethylamine and an alkali metal iodide in acetone under pressureat a temperature between about room temperature and about 75 0., (g)detritylating the iodide of the lit-choline ester of N-trityl-L-glutamicacid by contacting with an aqueous lower alkanoic acid, and (h)isolating a non-toxic salt of a-L-glutamyl choline substantially free ofits 'y-isomer.

References Cited in the file of this patent UNITED STATES PATENTS2,776,923 Nishizawa Jan. 8, 1957 OTHER REFERENCES R. B. Angler et al.:J. Org. Chem, volume 21 (1956), pages 15403.

UNITED STATES PATENT @FFICE QETIFICATE QQECH Patent, Noe. 3 OO9 923November 21 196 Gaston Amiard et a1.

It is hereby certified "that error appears in the above numberedpatentrequiring' correction and "that the said Letters Patent shouldread as corrected belowo after "CHLORQETHYU' insert a closingparenthesis; line 5G fer C H O NCU reed C H O NCTL Column 0 line uLlglutglmyl read We u L glutamyl column 5 line l8 "16$ reed +1605 Cclumn3 line 3 Signed and sealed this 8th day of May 19620 (SEAL) Attest:

DAVID L. LADD ERNEST W. SWIDER Attesting Officer I Commissioner ofPatents

7. A PROCESS FOR THE PRODUCTION OF A NON-TOXIC SALT OFA-L-GLUTAMYL-CHOLINE SUBSTANTIALLY FREE OF ITS $-ISOMER COMPRISING THESTEPS OF (A) ESTERIFYING L-GLUTAMIC ACID WITH GLYCOL-MONOCHLOROHYDRIN INTHE PRESENCE OF GASEOUS HYDROCHLORIC ACID AT ELEVATED TEMPERATURES, (B)NEUTRALIZING THE HYDROCHLORIDE OF DI-(B''-CHLORETHYL) L-GLUTAMATE BYTREATMENT WITH A STRONG BASE SELECTED FROM THE GROUP CONSISTING OFTRIETHYLAMINE AND ALKALI METAL HYDROXIDES, (C) REACTING THEDI-(B''-CHLOROETHYL) L-GLUTAMATE WITH TRITYL CHLORIDE IN THE PRESENCE OFTERTIARY AMINE, (D) DISSOLVING THE DI-(B''-CHLOROETHYL) N-TRITYL-LI-GLUTAMATE IN METHANOL AND REACTING WITH SODIUM METHYLATE, (E) DISSOLVINGTHE A-(B''-CHLOROETHYL)-$-METHYL N-TRITYL-L-GLUTAMATE IN AN INERTORGANIC SOLVENT AND SAPONIFYING WITH AN ALKALI METAL HYDROXIDE, (F)QUATERNIZING THE A-(B''-CHLOROETHYL) ESTER OF N-TRITYL-L-GLUTAMIC ACIDBY REACTING WITH TRIMETHYLAMINE AND AN ALKALI METAL IODIDE IN ANANHYDROUS ORGANIC SOLVENT UNDER PRESSURE AT ELEVATED TEMPERATURES, (G)DETRITYLATING THE IODIDE OF THE A-CHOLINE ESTER OF N-TRITYL-L-GLUTAMICACID BY CONTACTING WITH AN AQUEOUS LOWER ALKANOIC ACID, (H) ANDISOLATING A NON-TOXIC SALT OF A-L-GLUTAMYL CHOLINE SUBSTANTIALLY FREE OFITS $-ISOMER.